Mitochondria sense and respond to calcium signals. The mitochondrial response is required for metabolism and other aspects of cell function and is likely to be central for several disease mechanisms activated by genetic impairments or environmental stress. During cytoplasmic [Ca2+] ([Ca2+]c) oscillations, mitochondrial Ca2+ sensing is ensured by local Ca2+ transfer from IP3 or ryanodine receptors to the mitochondrial Ca2+ uptake sites. The local communication has been proposed to occur at the sites where the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is closely associated with the mitochondria. In the past project period we and others have shown that the ER/SR-mitochondrial associations are supported by direct interorganellar physical links, referred as tethers. Furthermore, we and others have shown that at the sites of the ER-mitochondrial associations, mitochondria are exposed to at least 10-fold higher local [Ca2+] concentration than the global [Ca2+] c rise evoked by IP3-linked hormones. These results strengthen the concept that the ER-mitochondrial interface hosts a special structural arrangement. However, the miniature interface (<200nm diameter) is difficult to study with currently available approaches. Our first hypothesis is that mitochondrial Ca2+ sensing depends on IP3R localization, expression level, isoform variety, and activation-deactivation kinetics. We propose that IP3 receptors, SERCA pumps and VDACs are concentrated at the interface and form mutual interactions to present low background and high signal intensity for Ca2+ delivery to the mitochondria. To explore the molecular composition and function of the ER-mitochondrial contacts we have developed and will employ an array of novel molecular tools. Very recently, MICU1, and MCU have been identified as the first molecular components of the mitochondrial Ca2+ uniport but their function remains elusive. Our second hypothesis is that to control the Ca2+ transfer across the inner mitochondrial membrane, MICU1, an EF hand domain protein binds to MCU, the pore forming domain of the uniporter in a Ca2+ sensitive manner. Binding of MICU1 confers cooperativity to the Ca2+ uniport, a benefit of which is that at low [Ca2+] mitochondria excluded from Ca2+ handling and avoid Ca2+ overload. The third hypothesis is that the mitochondrial response to Ca2+ involves K+ influx and an ensuing increase in matrix volume. Mitochondrial matrix volume changes are central to the regulation of mitochondrial fusion, and provide a novel mechanism for the calcium signal to control mitochondrial fusion through. Fusion events, calcium and mitochondrial volume will be followed by fluorescence measurements of several novel reporters down to the level of individual organelles. Collectively, these studies will enhance the understanding of the fundamental mechanisms of mitochondrial calcium signaling and dynamics and will provide several molecular tools that will also facilitate the investigation of many other signaling paradigms.